A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.
Aims The purpose of this study was to evaluate the in vitro effects of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and a downregulator of intracellular reactive oxygen species (ROS), on high glucose-induced oxidative stress on tenocytes. Methods Tenocytes from normal Sprague-Dawley rats were cultured in both control and high-glucose conditions. Apocynin was added at cell seeding, dividing the tenocytes into four groups: the control group; regular glucose with apocynin (RG apo+); high glucose with apocynin (HG apo+); and high glucose without apocynin (HG apo–). Reactive oxygen species production, cell proliferation, apoptosis and messenger RNA (mRNA) expression of NOX1 and 4, and interleukin-6 (IL-6) were determined in vitro. Results Expression of NOX1, NOX4, and IL-6 mRNA in the HG groups was significantly higher compared with that in the RG groups, and NOX1, NOX4, and IL-6 mRNA expression in the HG apo+ group was significantly lower compared with that in the HG apo– group. Cell proliferation in the RG apo+ group was significantly higher than in the control group and was also significantly higher in the HG apo+ group than in the HG apo– group. Both the ROS accumulation and the amounts of apoptotic cells in the HG groups were greater than those in the RG groups and were significantly less in the HG apo+ group than in the HG apo– group. Conclusion Apocynin reduced ROS production and cell death via NOX inhibition in high-glucose conditions. Apocynin is therefore a potential prodrug in the treatment of diabetic tendinopathy. Cite this article: Bone Joint Res 2020;9(1):23–28.
Template Specificity and Subunits of RNA Polymerase from Asporogenous Mutants of Bncillrts subfilis. Cnn. 9. Biochenm. 52, 966-973. In order to investigate relations between template specificity of RNA polymerase and sporulation, RNA polymerase activities in partially purified preparations from various asporogenous mutants were measured with poly[d(A-T)] or DNA from phage PBS 1% as template. Results obtained suggest that morphological changes occurring during spomlation may not be tightly linked temporally to transcriptional events.Subunits of RNA polymerase from these mutants were analyzed by sodium dodecyli sulfatepolyacrylamide gel electrophoresis after purification by (NH*)iSOe precipitation, DEAE-cellulose chromatography, phosphweilulsse chromatography, and glycerol gradient centrifugation. Phenylmethylsulfonyl fluoride was present throughout the purification procedure to prevent proteolytic degradation. It was found that p and P' subunits were present in 1:B ratio in all preparations. In addition to 8, p" and a subunits. a protein having a molecular weight of 95 008 was found in enzyme preparations from a wild-type strain and stage EI mutants harvested at f3-f@. This protein was absent in stage 0 mutants and in all strains harvested in log phase. The enzyme containing this protein was eluted from ghosphocellulose column with 0.6 2kf KC1 rather than 8.35 M KCl, which eluted the enzyme without the 95 0490 dalton protein. Furthermore the enzyme with this protein showed a sedimentation coefficient higher than that of the enzyme without the 95 000 dalton protein. Nishimoto, W. & Takahashi, I. (1974) Template Specificity and Subunits of RNA Polymerase from Asporogenous Mutants of BaciEEus subtilis. Can. J . Biockem. 52, 966-97 3.Afin dYCtudier les relations entre la sporulation et la spCcificit6 de la RNA polymkrase v i s -h i s la matrice, nous avons mesuri 19activitC RWA poBym6rasique de preparations partieliemen purifites parir de divers mutants asporogknes lorsqu'on utilise comme matrice un copolym&re poly[d(A-T)] ou le DNA du phage PBS 15. L s rCsuItats obtenus sugghrent que les changements morphologiques awompagnant la sporulation ne somt probablement pas relies ttroitement dans le temps aux diverses phases de B a transcription.Les sous-unites des RWA polymCrases de ces mutants sont analyskes par Clectrophor&se sur gel de dodCcylsulfate de sodiumpolyacrylamide aprts purification par precipitation avec le (NH4)aSOe, chromatographie sur DEAE-cellulose, chromatographie sur phospho-celluIose et centrifugation en gradient de glydrol. M w de gr6venir la digradation prottolytique, nous avons ajoutt du fluorrare de ph6nylm6tkylsulfonyIe durant tout le processus de purification. Dans toutes les preparations, Ies sous-unit& p et p' sont prksentes dans un rapport 1: 1. En plus des sous-unites p, p' et a, ume grottine de 95 000 daltons est retrouvCe dans les prCparations enzymatiques d'une souche de type sauvage et des mutants de stade PI r6colt6s B t5-f@. Cette prottiwe est absente chez Bes mutants de stade 8 et dan...
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